Current Pharmaceutical Biotechnology - Volume 18, Issue 14, 2017

Background: In the present study, the potential of methanolic leaf extract of Taraxacum officinale plant as a function of bio-inspired green synthesis for the fabrication of silver nanoparticles (AgNPs) has been explored. Methods: The bio-reduction of aqueous silver nitrate (AgNO3) solution was confirmed by visually detecting the color change from pale yellow to blackish-brown. Maximum absorbance was observed at 420 nm due to the presence of characteristic surface Plasmon resonance of nano silver by UV-visible spectroscopy. The role of various functional groups in the bio-reduction of silver and chemical transformation was verified by Fourier transform infrared spectroscopy (FTIR). Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) predict the shape (rocky, flack type, ellipsoidal, etc.), size (68 nm) and elemental composition (Ag as a major constituent) of the biosynthesized AgNPs, respectively. Results: Transmission electron microscopy (TEM) analysis further corroborated the morphology of the AgNPs. Color mapping and atomic force microscopy (AFM) confirmed the nano-sized topography. The dynamic light scattering (DLS) showed the charge, stability, and size of the AgNPs. The generated AgNPs presented potential antibacterial activities against Gram-positive and Gram-negative bacterial strains including Staphylococcus aureus, Escherichia coli, and Haemophilus influenzae. The biosynthesized AgNPs also showed antiproliferative activity against MCF-7 breast cancer cell line in a dosedependent manner. Conclusion: In conclusion, results clearly indicate that biosynthesized AgNPs could be used as effective nano drug for treating infectious diseases caused by multidrug resistant bacterial strains in the near future.

Background: Alzheimer's disease (AD) is one of most serious threats to human beings, however, the treatment is hindered by blood-brain barrier and poor intra-brain cell selectivity. Methods: In this study, we developed a novel dual targeting drug delivery system by modification of NL4 peptide and apolipoprotein A-I (ApoA-I) onto dendrimer particles that may efficiently deliver siRNA into neuron cells to down-regulate BACE1 and inhibit Aβ formation. The constructed ANNP/ siRNA was approximately 79.26 nm with a spherical structure and a zeta potential of 3.53 mV. At N/P ratio of 10, the siRNA could be completely packaged into particles to avoid degradation by RNAase. Results: In vitro, the modification with ApoA-I considerably increased bEnd.3 cell uptake and NL-4 considerably increased PC12 cell uptake. As a result, ANNP/siRNA showed higher uptake in both the cells. In addition, ANNP/siRNA could efficiently penetrate through bEnd.3 monolayers, which was 2.4-fold higher than unmodified complexes. In PC12 cells, the ANNP/siRNA could escape from endosomes and transport into cytoplasm after 8 h incubation, resulting in 87.5% BACE1 gene knockdown capacity, which was better than PEI. Additionally, the particles showed low cytotoxicity to both bEnd.3 and PC12 cells. Conclusion: In conclusion, this study preliminarily demonstrated that ApoA-I and NL4 dual modified dendrimer nanoparticles were efficient carriers for siRNA delivery to AD bearing brain.

Background: Ethanolic extract of G. procumbens leaves has been previously shown to possess antihyperlipidemic effects. Objective: This study was designed to prepare caffeoylquinic acids rich and poor fractions of the ethanolic extract using resin column technology and compare their antihyperlipidemic and antioxidant potentials. Results: Among the treatment groups, caffeoylquinic acids rich fraction (F2) and chlorogenic acid (CA, one of the major caffeoylquinic acids) showed potent antihyperlipidemic effects, with significant reductions in total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C), very low-density lipoprotein-cholesterol (VLDL-C), atherogenic index (AI) and coronary risk index (CRI) (p<0.01 or better) compared to the hyperlipidemic control at the 58 h. The effect was better than that of ethanolic extract. In addition, only F2 significantly increased the high-density lipoproteincholesterol (HDL-C) level (p<0.05). F2 showed better effect than CA alone (60 mg) despite the fact that it only contained 9.81 mg CA/1000 mg dose. The findings suggest that the di-caffeoylquinic acids (86.61 mg/g dose) may also in part be responsible for the potent antihyperlipidemic effect shown by the F2. Likewise, F2 showed the highest antioxidant activity. Thus, simple fractionation of ethanolic extract using the Amberlite XAD-2 resin technique had successfully enriched the caffeoylquinic acids into F2 with improved antihyperlipidemic and antioxidant capacities than that of the ethanolic extract. Conclusion: The resin separation technology may find application in caffeoylquinic acids enrichment of plant extracts for pre-clinical studies. The F2 has potential for development into phytopharmaceuticals as adjunct therapy for management of hyperlipidemia.

Background: Drug-induced myopathy is a serious side effect that often requires removal of a medication from a drug regimen. For most drugs, the underlying mechanism of drug-induced myopathy remains unclear. Monocarboxylate transporters (MCTs) mediate L-lactic acid transport, and inhibition of MCTs may potentially lead to perturbation of L-lactic acid accumulation and muscular disorders. Therefore, we hypothesized that L-lactic acid transport may be involved in the development of drug-induced myopathy. The aim of this study was to assess the inhibitory potential of 24 acidic drugs on L-lactic acid transport using breast cancer cell lines Hs578T and MDA-MB-231, which selectively express MCT1 and MCT4, respectively. Methods: The influx transport of L-lactic acid was minimally inhibited by all drugs tested. The efflux transport was next examined: loratadine (IC50: 10 and 61 μM) and atorvastatin (IC50: 78 and 41 μM) demonstrated the greatest potency for inhibition of L-lactic acid efflux by MCT1 and MCT4, respectively. Acidic drugs including fluvastatin, cerivastatin, simvastatin acid, lovastatin acid, irbesartan and losartan exhibited weak inhibitory potency on L-lactic acid efflux. Results: Our results suggest that some acidic drugs, such as loratadine and atorvastatin, can inhibit the efflux transport of L-lactic acid. Conclusion: This inhibition may cause an accumulation of intracellular L-lactic acid leading to acidification and muscular disorders.

Background: Drug transporters function as gatekeepers and modulate drug access into body and various tissues. Thus, a thorough and precise understanding of transporter liability for compound uptake and efflux is critical during drug development. Methods: In the present study, we assessed the apparent permeability (Papp) and compared efflux ratio of various compounds in stably transfected Madin-Darby Canine Kidney (MDCKII) cells overexpressing human P-gp (MDCKII-MDR1), human BCRP (MDCKII-BCRP), wild-type (MDCKII-WT), and Caco-2 cell monolayers. Results: We observed that quinidine, a substrate for MDR1 transporter, showed efflux ratio (Papp B-A/ Papp A-B) of 838 in MDCKII-MDR1 cells which plummeted to 14 in presence of verapamil, a known inhibitor of MDR1. With MDCKII-WT cells, Papp of quinidine dropped from 2 to 1, in the presence of verapamil. Caco-2 cells showed a diminutive decrease in efflux ratio of quinidine from 2.5 to 1.6 by verapamil. Prazosin and dantrolene were evaluated in MDCKII-BCRP cells and were found to have 80-fold higher efflux ratio compared to MDCKII-WT cells. In Caco-2 cells, prazosin and dantrolene showed efflux ratio of 4 and 2, respectively. Rhodamine-123, a fluorogenic probe substrate of MDR1 showed an efflux ratio of 4 in Caco-2 cells and BCRP substrate estrone-3-sulphate showed an efflux ratio of 7. In presence of BCRP inhibitor fumitremorgin-c, the efflux ratio of estrone-3-sulfate dropped to 1 in Caco-2 cells. Conclusion: The very high efflux ratios of MDR1 and BCRP substrates in transfected MDCKII cells clearly demonstrate the potential usefulness of these models to provide more definitive data to evaluate the transporter involvement compared to Caco-2 or MDCKII-WT cells.

Background: Expression vector is an important component in the production of therapeutic recombinant proteins. Most of the commercialized expression vectors apply antibiotic-based selection system. Meanwhile, World Health Organization highly recommends for the alternative system due to its potentials to cause spreading of resistance gene and hypersensitivity to some people. Methods: In current work, we developed an expression system for Escherichia coli using the toxinantidote system in two separated plasmids. An antidote gene (ccdA) with its natural promoter and terminator was constructed in a plasmid (pDCSAsod) containing a DNA fragment encoding recombinant superoxide dismutase from Staphylococcus equorum (rMnSODSeq) as a model. The gene expression was directed under T7 promoter and regulated by lac operator. The toxin gene (ccdB) was located in a separate plasmid (pDCSB) under PBAD promoter. This study aimed to study the growth profile of the host in the presence of both plasmids, to determine plasmids stability, and the effect of the toxinantidote system on rMnSODSeq production and activity. Results: The presence of both plasmids did not affect the growth profile of E. coli BL21(DE3), while the plasmid stability was 94% for pDCSAsod and 68% for pDCSB at the end of protein production time. The yield of purified rMnSODSeq was 3.2 mg/ml and the enzyme was shown to be active by a zymography assay. Conclusion: In conclusion, for the first time, we show that toxin-antidote system in two separated plasmids has the potential for the production of recombinant therapeutic proteins and is more flexible in choosing the E. coli strain compared to established chromosomally integrated toxin-antidote selection system.

Background: The aim of this work was to compare water and organic extracts, infusions and tinctures from flowers and leaves of Calendula officinalis in terms of their biological activity and composition. The purpose of work was investigation whether the leaves and stems are really the waste or they contain interesting substances which could be utilized. Antimicrobial, antifungal, antioxidant and anti-inflammatory activities were studied. Then, the ability to inhibit collagenase was studied as well. Cytotoxicity was tested for all the samples on mammalian cell lines. Methods: To determine the composition of extracts, infusions and tinctures phytochemical analysis (the set of colour reactions for the detection of groups of biologically active compounds) was carried out and showed that samples from flowers and leaves contain the same groups of biologically active substances (proteins and amino acids, reducing sugars, flavonoids, saponins, phenolics, terpenoids, steroids, glycosides). The antimicrobial activity of tested samples was proved, where the most sensitive bacterium was Micrococcus luteus and the most sensitive yeast was Geotrichum candidum. Results: The study of anti-collagenase activity has shown that the enzymatic reaction of collagenase was affected by all tested samples and their effect was concentration dependent. Cytotoxicity of water and methanol extracts at cell lines HEK 293T and HepG2 was observed. Conclusion: Cells HepG2 were more sensitive than cells HEK 293T. Using cell line RAW 264.7, antiinflammatory activity of all samples was observed. Tincture of leaves was the most effective.

Background: Excessive alcohol consumption damages the intestine and liver cells directly as well as through unbalancing the gut microbiota. Objective: The current study was undertaken to correlate the alcohol consumption and change in urinary metabolites profile linked with gut microbiota. Method: Non-alcoholic (control) healthy (n=22) and moderate alcoholic (n=26) males with an average age of 39.3±1.83 years subjected to alcohol use disorders identification test (AUDIT) were considered for study. First pass urine and blood samples were collected in the morning. Results: Liver function test showed the increased levels of γGT, AST and ALT to 40.3 ± 2.3, 53.3 ± 0.7, and 38.9 ± 0.5 U/L, respectively. Urine samples were processed and subjected to HPLC-Q-TOFMS analysis in positive and negative ion polarity modes. Mass data were processed to align and filter out insignificant entities and subjected to One-way ANOVA with Bonferroni multiple testing corrections analysis. The analysis provided list of 211gut microbes specific metabolites with p>0.05 and fold change >1.5. All metabolites were identified using standards and referring to METALIN library of standard metabolites. Further analyses showed that alcohol intake disturbed more than ten metabolic pathways. Tryptophan, tyrosine, branched chain amino acids and short-chain fatty acids metabolism were the significantly disturbed pathways in alcoholics. Conclusion: Correlation of various metabolites with gut microbiota showed that chronic and moderate dose intake of alcohol decreased the level of Bifidobacterium, Lactobacillus Ruminococcus and Faecalibacterium spp. and increased the levels of Proteobacteria, Alcaligenes and Clostridium.

Background: Aiming to improve the laser-induced forward transfer (LIFT) cell isolation process, a polydimethylsiloxane (PDMS) layer with micro-hole arrays was employed to improve the cell separation precision, and a microchip with heater was developed to maintain the working area at 100% humidity and 37°C with the purpose to preserve the viability of the isolated cells. Methods: A series of experiments were conducted to verify the contributions of the optimization to LIFT cell isolation process as well as to study the effect of laser pulse energy, laser spot size and the titanium thickness on cell isolation. With 40μm laser spot size and 40nm thick of titanium, laser energy threshold for 100% single cell isolating succeed ratio is 7μJ. Results: According to the staining images and proliferation ratios, the chip did help to improve the cell availability and the cells can recover from the juries at least a day earlier comparing to the samples processed without the chip. Conclusion: With a Lattice Boltzmann model, the cell isolation process is numerically studied and it turns out that the micro-hole makes the isolation process shift to a micro-syringe injection model leading to the lower laser energy threshold for cell separation and fewer injuries.